Nucleic acid analysis device, method for producing same, and nucleic acid analyzer

ABSTRACT

Disclosed is a technique for binding microparticles to patterned bonding pads of a metal (e.g., gold) formed on a support. The microparticles each carry a nucleic acid synthetase or DNA probe immobilized thereon for capturing a nucleic acid sample fragment. The technique involves forming, on a support surface, a film having a thickness equivalent to that of the bonding pads; controlling the size of microparticles with respect to the size of bonding pads; and thereby immobilizing microparticles each bearing a single nucleic acid sample fragment to the bonding pads in a one-to-one manner in a grid form. This allows high-density regular alignment and immobilization of many types of nucleic acid fragment samples on a support and enables high-throughput analysis of nucleic acid samples. Typically, immobilization of microparticles at 1-micrometer intervals easily provides a high density of 10 6  nucleic acid fragments per square millimeter.

TECHNICAL FIELD

The present invention relates to a nucleic acid analysis device, aproduction method thereof, and a nucleic acid analyzer using the same.

BACKGROUND ART

New techniques have been developed as nucleic acid analysis devices fordetermining base sequences of DNA (deoxyribonucleic acid) or RNA(ribonucleic acid). A method utilizing electrophoresis, which is now ingeneral use, involves preparing beforehand a cDNA (complementary DNA)fragment sample synthesized through a reverse transcription reaction ofa DNA fragment or an RNA sample for sequence determination; performing adideoxy chain termination reaction by well-known Sanger's sequencingmethod; thereafter performing electrophoresis for the sample; andmeasuring a pattern of separation and development of molecular weight toanalyze the pattern.

Independently, there has been recently developed a technique ofimmobilizing many DNA sample fragments as samples on a support so as todetermine information on sequences of the many fragments in parallel. ADNA sequencer using this technique is referred to as a massivelyparallel sequencer. The massively parallel sequencer performs DNAelongations on fluorescence-labeled bases as a substrate in parallel athundreds of thousands to millions of points and detects fluorescence ofreacted bases to determine a DNA nucleotide sequence. Such massivelyparallel sequencers are categorized as those according to acluster-basis process and those according to a single-molecule-basisprocess. The respective processes will be described below.

Initially, the cluster-basis process will be described. Thecluster-basis process involves analysis of clusters each of which is anamplified DNA and is in the form of a bundle of DNAs. Typically,Non-Patent Literature (NPL) 1 describes a technique including: preparingmicroparticles bearing DNA fragments, performing polymerase chainreactions (PCRs) on the microparticles to amplify DNA fragments intomany copies, and placing the microparticles bearing PCR-amplified DNAfragments in a plate having many wells, followed by pyrosequencing-basedreading. The wells each have an opening with a size equal to the size ofeach of the microparticles.

NPL 2 describes a technique including: preparing microparticles bearingDNA fragments, performing polymerase chain reactions on themicroparticles, scattering the microparticles onto a glass support,immobilizing the microparticles thereto, performing enzymatic reactions(ligation reactions) on the glass support to allow the DNA fragments toincorporate a substrate having a fluorescent dye thereinto, detectingfluorescence emitted from the fluorescent dye, and thereby obtaininginformation on nucleotide sequence of each of the fragments.

Next, the single-molecule-basis process will be described. Thesingle-molecule-basis process includes: hybridizing a labeled nucleicacid with a probe without amplification, and identifying a nucleotidesequence while elongating the nucleic acid one base by one base withnucleotides each having a fluorescent dye. This technique is reported inNPL 3. Typically, the technique described in NPL 3 is a technique ofpreparing a plate having many wells and arranging a nucleic acidsynthetase on the plate. In this technique, fluorescence is detectedwhile allowing nucleotides having a fluorescent dye to be incorporatedinto a nucleic acid to thereby elongate the nucleic acid, and thusinformation on nucleotide sequence of each fragment is obtained.

CITATION LIST Non-Patent Literature

-   NPL 1: Nature 2005, Vol. 437, pp. 376-380-   NPL 2: Genome Research 200, Vol. 18, pp. 1051-1063-   NPL 3: Science 2009, Vol. 323, pp. 133-138-   NPL 4: Nanotechnology, 2007, Vol. 18, pp. 044017-044021.-   NPL 5: P.N.A.S. 2006, Vol. 103, pp. 19635-19640

SUMMARY OF THE INVENTION Technical Problems to be Solved by theInvention

Of massively parallel sequencers, those according to the cluster-basisprocess analyze DNA clusters as bundles of amplified DNAs, whereas thoseaccording to the single-molecule-basis process directly analyze DNAswithout amplification. The single-molecule-basis process does notrequire an amplification process and can save process and running cost.The cluster-basis process is limited in base length to be read due to adephasing phenomenon, in which sequencing reactions among pluralamplified DNAs occur at different times. By contrast, thesingle-molecule-basis process does not theoretically undergo dephasingand indicates the possibility that bases in a significantly longerlength can be read. This requires, however, a technique of immobilizinghundreds of thousands of nucleic acid sample fragments to a support on asingle molecule basis or on a group of molecules basis. In thistechnique, maximally regular immobilization of nucleic acid samples to aflat, smooth support is desired. This is because random immobilizationof microparticles bearing nucleic acid samples and being scattered on aflat, smooth support can be easily performed, but, upon reading ofsequences by fluorometry, it takes an extremely long time to processnumerical data, which data have been obtained from images of themicroparticles being present at random, where the images are detectedwith a charge coupled device (CCD) camera.

Accordingly, an object of the present invention is to solve theaforementioned problems.

Solution to Problems

The present invention provides, in an aspect, a nucleic acid analysisdevice which comprises:

a support; a plurality of bonding pads arranged on a surface of thesupport;a thin-film layer covering the surface of the support in a region otherthan the bonding pads;microparticles, each of the microparticles is bound to each of thebonding pads; anda probe molecule or molecules of a single type immobilized on each ofthe microparticles,in which the microparticles are bound to the bonding pads throughchemical bonds, and the thin-film layer is capable of suppressingnon-specific adsorption of the microparticles on the surface of thesupport.

The present invention provides, in another aspect, a method forproducing a nucleic acid analysis device which comprises the steps of:

forming a thin metal film on a surface of a support;selectively etching the thin metal film to form a plurality of bondingpads;introducing a linear molecule film into each of the bonding pads, thelinear molecule film capable of being adsorbed on the bonding pads;introducing microparticles onto the linear molecule film and binding themicroparticles to each of the bonding pads through a chemical bond; andimmobilizing a probe molecule or molecules to each of the microparticlesthrough a chemical bond.

The present invention provides, in yet another aspect, a method forproducing a nucleic acid analysis device which comprises the steps of:

forming a thin metal film on a surface of a support;introducing a linear molecule film onto the thin metal film, the linearmolecule film capable of being adsorbed on the thin metal film;selectively etching the thin metal film and the linear molecule film andforming a plurality dummy bonding pads;removing the dummy bonding pads to expose the linear molecule film toform a plurality each of the bonding pads having the thin metal film andthe linear molecule film;binding microparticles to each of the exposed linear molecule filmsthrough a chemical bond; andimmobilizing a probe molecule or molecules to each of the microparticlesthrough a chemical bond. As used herein the term “dummy bonding pad”corresponds to an etching mask which has a shape equivalent to that ofan actual bonding pad and is formed upon patterning of a thin metal filmon a linear molecule film that binds the microparticles to the thinmetal films. A thin film for the suppression of non-specific adsorptionof the microparticles is formed on the etching mask (dummy bonding pad),but the thin film on the etching mask will be removed together with theetching mask upon binding of the microparticles to the bonding pads.

The present invention provides, in still another aspect, a nucleic acidanalyzer, which comprises:

a nucleic acid analysis device including a support and microparticlesregularly immobilized on the support, the microparticles each of whichhas the probe molecule or molecules capable of capturing a nucleic acidto be analyzed;a supplier for supplying a nucleic acid sample and a nucleotide having afluorescent dye to the nucleic acid analysis device; an irradiator forirradiating the nucleic acid analysis device with light; andan emission detector for detecting fluorescence emitted from thefluorescent dye incorporated into a nucleic acid chain through nucleicacid elongation induced by the coexistence of a nucleotide, a nucleicacid synthetase, and a nucleic acid sample. The nucleic acid analysisdevice, which comprises:a plurality of bonding pads arranged on the surface of the support atpositions where the microparticles are immobilized;a thin film layer covering the surface of the support in a region otherthan the bonding pads;microparticles, where a single microparticle is bound to each of thebonding pad; anda probe molecule or molecules of a single type immobilized on each ofthe microparticles,in which the microparticles are bound to the bonding pads throughchemical bonds, and the thin film layer is capable of suppressingnon-specific adsorption of the microparticles on the surface of thesupport.

Advantageous Effects of Invention

The present invention allows microparticles to be reliably arranged in adesired alignment and immobilized on many bonding pads in a nucleic acidanalysis device and thereby enables high-precision analysis of nucleicacids with less noise.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view illustrating an exemplary configuration ofa nucleic acid analysis device according to an embodiment of the presentinvention.

FIG. 2 is a cross-sectional view illustrating a partial configuration asone embodiment of the nucleic acid analysis device illustrated in FIG.1.

FIG. 3 is a cross-sectional view illustrating a partial configuration asanother embodiment of the nucleic acid analysis device illustrated inFIG. 1.

FIG. 4A is a schematic cross-sectional view illustrating an exemplaryrelationship between the dimensions of a bonding pad formed on a supportand the diameters of beads (microparticles) arranged on the bonding pad.

FIG. 4B is a schematic cross-sectional view illustrating anotherexemplary relationship between the dimensions of a bonding pad formed ona support and the diameters of beads arranged around the bonding pad.

FIG. 4C is a schematic cross-sectional view illustrating still anotherexemplary relationship between the dimensions of a bonding pad formed ona support and the diameter of a bead arranged on the bonding pad.

FIG. 5 is a schematic cross-sectional view illustrating an exemplaryrelationship between the dimensions of a bonding pad formed on a supportand the diameter of a bead arranged on the bonding pad, according to anembodiment of the present invention.

FIG. 6 is a schematic cross-sectional view illustrating an exemplaryrelationship between the dimensions of a bonding pad formed on a supportand the diameter of a bead arranged on the bonding pad, according toanother embodiment of the present invention.

FIG. 7 is a flowchart illustrating a method for producing a nucleic acidanalysis device according to an embodiment of the present invention.

FIG. 8 is a flowchart illustrating a method for producing a nucleic acidanalysis device according to another embodiment of the presentinvention.

FIG. 9 is a diagram illustrating an exemplary configuration of a nucleicacid analyzer including a nucleic acid analysis device according to thepresent invention.

DESCRIPTION OF EMBODIMENTS

The present inventors made intensive investigations on techniques fordensely and regularly aligning and immobilizing nucleic acid samplefragments one by one to a support. As a result, they have found atechnique for immobilizing nucleic acid sample fragments to a supportone by one. This technique includes allowing microparticles tospecifically react with a plurality of (preferably a multiplicity of)patterned bonding pads of a metal (such as gold) on a one-to-one basis,in which each of the microparticles have a nucleic acid synthetase orDNA probe immobilized thereto and capable of capturing a nucleic acidsample fragment. As used herein the term “one-to-one basis” refers tothat a probe molecule or molecules of a single type are allowed tospecifically react with one microparticle; and the number of probemolecule or molecules is not limited, as long as they are of a singletype.

The present invention provides, in an embodiment, a nucleic acidanalysis device which includes a support and microparticles regularlyimmobilized on the support, in which each of the microparticles have aprobe molecule capable of capturing a nucleic acid to be analyzed,

in which the nucleic acid analysis device further includes bonding padson the support at positions where the microparticles are immobilized;the microparticles are bound to the bonding pads through chemical bonds;the nucleic acid analysis device further includes a thin-film layer onthe surface of the support, and the thin-film layer is capable ofsuppressing or inhibiting non-specific adsorption of the microparticles;a part of each of the bonding pad is exposed from the thin-film layer;and each of the bonding pad, except for the part, is covered with thethin-film layer. The bonding pads are preferably regularly arranged onthe support. Two or more probe molecules may be immobilized on a singlemicroparticle.

More specifically, each of the microparticles bearing a substanceimmobilized thereto are prepared, which substance is capable ofcapturing a nucleic acid sample fragment and is typified by nucleic acidsynthetases and DNA probes, and the microparticles are bound to bondingpads on a support, in which bonding pads include a metal such as goldand are formed in a pattern. In this process, a thin film is formed onthe support, which thin film includes, for example, an organic polymerand advantageously prevents non-specific adsorption of themicroparticles; the thin film of the organic polymer is designed to havea thickness equivalent to that of the bonding pads; and the sizes of thebonding pads is controlled with respect to the sizes of themicroparticles. These can remarkably increase the percentage ofmicroparticles immobilized on bonding pads in a one-to-one manner,thereby the microparticles capture and bear a single molecule of nucleicacid sample fragment.

The present invention further provides, in another embodiment, a nucleicacid analysis device which includes a support and microparticlesregularly immobilized on the support, in which each of themicroparticles have a probe molecule capable of capturing a nucleic acidto be analyzed; the nucleic acid analysis device further includesbonding pads on the support at positions where the microparticles areimmobilized; the microparticles are bound to the bonding pads throughchemical bonds; the nucleic acid analysis device further includes athin-film layer on the support; the thin-film layer is capable ofpreventing non-specific adsorption of the microparticles; apart of eachbonding pads is exposed from the thin-film layer; and each of thebonding pads, except for the part, is covered with or embedded in thethin-film layer.

A single molecule or two or more molecules for the probe molecule may beimmobilized on a single microparticle in the nucleic acid analysisdevice. Two or more probe molecules, when immobilized on the singlemicroparticle, are identical (of a single type).

The probe molecule in the nucleic acid analysis device may be a nucleicacid or a nucleic acid synthetase. The microparticles in the nucleicacid analysis device may include a material selected from the groupconsisting of semiconductors, metals, inorganic polymers, and organicpolymers. The bonding pads preferably include a material selected fromthe group consisting of gold, titanium, nickel, and aluminum. The eachof the bonding pads preferably have an apparent diameter of one time orless that of the microparticles.

A multiplicity (thousands to hundreds of thousands) of the bonding padsare preferably regularly arranged on the support. In an embodiment, twoor more probe molecules may be immobilized on a single microparticle.This embodiment enables more simple control of the number of probes andeasier production of a nucleic acid analysis device than an embodimentin which a single probe molecule is immobilized on a singlemicroparticle. The each of the bonding pads preferably have an apparentdiameter one time or less that of the microparticles.

The present invention will be illustrated with reference to severalspecific embodiments below. Specifically, in an embodiment, disclosed isa nucleic acid analysis device which includes a support andmicroparticles regularly immobilized on the support, in which each ofthe microparticles have a probe molecule capable of capturing a nucleicacid to be analyzed, the nucleic acid analysis device further includesbonding pads on the support at positions where the microparticles areimmobilized, and the microparticles are bound to the detection pads(bonding pads) through chemical bonds.

In another embodiment, disclosed is a nucleic acid analyzer whichincludes a device for selectively obtaining only each of themicroparticles having a single probe molecule; a nucleic acid analysisdevice including a support and the microparticles regularly immobilizedon the support; a supplier for supplying a nucleic acid sample and anucleotide having a fluorescent dye to the nucleic acid analysis device;an irradiator for irradiating the nucleic acid analysis device withlight; and an emission detector for measuring fluorescence emitted fromthe fluorescent dye incorporated in a nucleic acid chain due to nucleicacid elongation induced by the coexistence of a nucleotide, a nucleicacid synthetase, and a nucleic acid sample on the nucleic acid analysisdevice. The nucleic analyzer obtains information on nucleotide sequenceof the nucleic acid sample.

In yet another embodiment, the nucleic acid analysis device may furtherinclude bonding pads on the support at positions where themicroparticles are immobilized, the microparticles are bound to thebonding pads through chemical bonds, the each of the bonding pads have adiameter equal to or less than that of the microparticles, the nucleicacid analysis device further includes a thin-film layer on the support,the thin-film layer including an organic polymer as a material, a partof each bonding pad is exposed from the thin-film layer, and eachbonding pad, except for the part, is covered with the thin-film layer.

In an embodiment, a single molecule or two or more molecules of theprobe molecule are immobilized on a single microparticle. Two or moreprobe molecules, when immobilized on a single microparticle, should beof the same type. Probe molecules of different types, if present on asingle microparticle, give different signals, and this may impedeanalysis.

The microparticles may include a material selected from the groupconsisting of semiconductors, metals, inorganic polymers, and organicpolymers. These materials may be spherical or non-spherical.

The bonding pads may include a material selected from the groupconsisting of gold, titanium, nickel, and aluminum. The smallerthicknesses the bonding pads have, the better. The bonding pads, ifhaving a thickness more than a specific upper limit, could capture twoor more microparticles. The bonding pads may have an arbitrary planarshape, such as a circular, square, rectangular, or elliptical shape.

The microparticles for use in the present invention onto which probemolecules are immobilized are often spherical or spheroidal, but are notalways spherical and may include amorphous or polygonal particles. Thebonding pads to which the microparticles are immobilized need not bespherical. For these reasons, the sizes of the microparticles (spheres)and of the bonding pads (films) are each indicated by an apparentaverage diameter. Typically, when having a major axis D₁ and a minoraxis D₂, the microparticle has an apparent average diameter D of(D₁+D₂)/2. When D₁ is equal to D₂, D is equal to D₁ or D₂. Likewise,when having a rectangular, elliptical, or another planar shape andhaving a long-side length L₁ and a short-side length L₂, the bonding padhas an apparent average diameter L of [(L₁ ²+L₂ ²)]^(1/2). When having asquare planar shape, the bonding pad has an apparent average diameter Lof the square root of (2L₁) or the square root of (2L₂). When having aperfectly circular or a similar planar shape, the bonding pad has anapparent average diameter L of equal to L₁ or L₂.

The microparticles have sizes (apparent average diameters) of preferably1 nm to 200 nm, and particularly preferably 5 to 100 nm. The bondingpads have sizes (apparent average diameters) of preferably twice orless, and particularly preferably one time or less the diameters of themicroparticles. Though not critical, the bonding pads have thicknessesof preferably 1 nm to 100 nm, and particularly preferably 3 to 50 nm. Asused herein the term “diameter (s)” of microparticles and of bondingpads refers to size(s) of the microparticles and of the bonding padseven when the microparticles and the bonding pads include those beingnot spherical or not having a perfectly circular planar shape, asmentioned above.

The bonding pads have diameters L of preferably twice or less, and morepreferably one time or less the diameters of the microparticles. Whenbeing non-circular or elliptical, the bonding pads have major axes ofpreferably twice or less the apparent diameters of the microparticles.

The above and other novel features and advantageous effects of thepresent invention will be illustrated below with reference to theattached drawings.

The present invention will be illustrated in detail with reference tospecific embodiments thereof below, for complete comprehension of theinvention. It should be noted, however, that the description below isnever construed to limit the scope of the present invention.

First Embodiment

A structure of a device according to the first embodiment will bedescribed with reference to FIGS. 1 and 2. Bonding pads 102 areregularly arranged on a flat (smooth) support 101. The each bonding pads102 have dimensions of a thickness of 10 nm and a diameter of 40 nm andmay be regularly arranged typically in a grid array as illustrated inFIG. 1. This allows the bonding pads to be aligned at uniform intervals.Noise could come into signals if the bonding pads partially excessivelyapproach to each other.

The bonding pads 102 are bound via linear molecules 105 to themicroparticles 103 through chemical bonds. A terminal functional group106 of each linear molecule 105 is preferably bound to a bonding pad 102through chemical interaction. In this case, the functional group 106preferably weakly interacts with the flat, smooth support 101 butstrongly interacts with the bonding pad 102. From this viewpoint, aquartz glass, sapphire, or silicon support may be used as the flat,smooth support.

The bonding pad 102 may include a material selected from the groupconsisting of gold, titanium, nickel, and aluminum. The functional group106 for use herein may be chosen in consideration of the combination ofthe flat, smooth support 101 and the bonding pad 102 and may be selectedtypically from sulfhydryl group, amino group, carboxyl group, phosphoricgroup, and aldehyde group. The linear molecule 105 plays a role ofbinding the microparticle 103 and the bonding pad 102 to each other, isnot significantly limited in length, but is preferably a linear(straight-chain) molecule having about three to about twenty carbonatoms in terms of length.

A terminal functional group 107 of the linear molecule 105 causesadhesion between a microparticle 103 and the linear molecule. A thinfilm 108 is arranged on the flat, smooth support. The thin film 108 iscapable of suppressing non-specific adsorption. The thin film 108preferably has a thickness equivalent to that of a bonding pad 102 so asto fully cover the lateral side of the bonding pad 102. The thin film108 preferably includes, as a material, an organic polymer capable ofpreventing non-specific adsorption of the microparticle 103. Exemplaryorganic polymers for use herein include polyethylene glycols (PEGs),polyacrylamides, and 3-glycidoxypropylmethoxy silane (COPS).

Exemplary microparticles 103 for use herein include metalmicroparticles, semiconductor microparticles, inorganic polymermicroparticles, and organic polymer microparticles. Typically, goldmicroparticles having a diameter of 5 nm to 100 nm are commerciallyavailable and are usable herein. Semiconductor microparticles asmicroparticles of a compound semiconductor, such as CdSe, havingdiameters of about 10 nm to about 20 nm are commercially available andare usable herein.

Fluorescent emission can be enhanced for observation by using, as themicroparticles, microparticles such as gold, silver, platinum, oraluminum microparticles having diameters of about 100 nm or less,because such microparticles can induce localized plasmon excitation at awavelength within the visible region. For example, fluorescenceenhancement by surface plasmon of gold microparticles is reported inNanotechnology, 2007, vol. 18, pp. 044017-044021 (NPL 4). This enablesenhancement of fluorescence from a fluorescent dye bound to a nucleotidefor fluorescent detection and increases the signal-to-noise (S/N) level.Particularly when a nucleic acid synthetase is used as the probemolecule 104, a fluorescent dye can be continuously introduced into theelectric field enhanced by localized-plasmon, and this advantageouslyenables stable fluorescence enhancement.

When semiconductor microparticles are used as the microparticles, theobservation of fluorescence from the fluorescent dye bound to eachnucleotide may be performed by exciting the semiconductor microparticleswith light from an external light source and transferring the excitationenergy to a fluorescent dye bound to the incorporated nucleotide. Inthis case, the excitation advantageously requires only a single type oflight source, because it is enough for an excitation light source toexcite only the semiconductor microparticles. Typically, microparticleshaving diameters of 15 to 20 nm (product name: “Qdot® streptavidinconjugate” (Invitrogen (Life Technologies Corporation))) may be used.

The inorganic polymer microparticles and organic polymer microparticlesare available also as commercial products such as microparticlesmodified in physical properties such as density, particle size, andelectric charge density; microparticles imparted with chemicalproperties typically by the action of a functional group or spacer; andmicroparticles labeled typically with a biomolecule such asstreptavidin.

Inorganic polymer microparticles, when employed, may be commerciallyavailable. Typically, silica microparticles having diameters of 30 to500 nm are commercially available as inorganic polymer microparticles.They are typified by silica microparticles such as Sicastar®,amino-labeled (micromod Partikeltechnologie GmbH) having diameters of 30to 500 nm; and Sicastar®, streptavidin-labeled (micromodPartikeltechnologie GmbH) having diameters of 100 to 500 nm.

Organic polymer microparticles may be available as commercial productssuch as latex microparticles having diameters of 15 to 500 nm, which aretypified by Micromer®, amino-labeled (micromod Partikeltechnologie GmbH)having diameters of 15 to 500 nm; and Micromer®, streptavidin-labeled(micromod Partikeltechnologie GmbH) having diameters of 100 to 200 nm,each as latex microparticles. Such amino-labeled microparticles ofinorganic polymer or organic polymer may be modified with avidin byreacting microparticles sequentially with biotin-succinimide(NHS-Biotin; Pierce Biotechnology, Inc.) and with streptavidin in thisorder, as with gold or platinum microparticles. When an oligonucleotideis used as a nucleic-acid-capturing probe 210, the oligonucleotide maybe synthesized via terminal modification with biotin. The resultingoligonucleotide can be readily immobilized on a microparticle.

When a nucleic acid synthetase is used as a nucleic-acid-capture probe210, an expression system may be first established using the RTS AviTagE. coli biotinylation kit (Roche Applied Science (Roche DiagnosticsCorporation)) to produce a nucleic acid synthetase. The produced nucleicacid synthetase can be readily immobilized on a microparticle.Functional groups usable as the functional group 107 may differdepending on the type of microparticles. For example, when goldmicroparticles are used, sulfhydryl group or amino group is preferred.When semiconductor microparticles, inorganic polymer microparticles, ororganic polymer microparticles are used, commercially availablemicroparticles with surfaces modified with streptavidin may be used. Inthis case, biotin can be used as the functional group 107.

The probe molecule 104 for capturing a nucleic acid may be asingle-stranded nucleic acid molecule such as DNA or RNA. One end of thenucleic acid molecule may be previously modified in the above manner aswith the functional group 107 to allow the nucleic acid molecule toreact with a microparticle 103. A nucleic acid binding protein ornucleic acid synthetase can also be used as the probe molecule 104 forcapturing a nucleic acid. A nucleic acid binding protein or nucleic acidsynthetase, when synthetically prepared using a specific reagent, can bereadily immobilized on the surface of a semiconductor microparticlemodified with a commercially available biotin. The specific reagent is areagent for introducing an avidin tag into an expressed protein and iscommercially available. A single-stranded nucleic acid molecule, whenused as a probe molecule 104 for capturing a nucleic acid, can capture asample nucleic acid molecule having a specific complementary sequence.In an embodiment illustrated in FIG. 2, a single probe molecule 104 isimmobilized on a single microparticle 103. However, two or more probemolecules may be immobilized on a single microparticle as illustrated inFIG. 3. Even in this case, however, the two or more probe moleculesshould be of the same type.

Supply of a nucleic acid synthetase or a nucleotide after the capture ofthe nucleic acid can induce nucleic acid elongation on the support.Likewise, a nucleic acid binding protein, when supplied, can capture anucleic acid having a specific sequence. A nucleic acid synthetase, whenused as the probe molecule 104, could capture a nonspecific samplenucleic acid molecule. Also in this case, supply of nucleotides caninduce nucleic acid elongation on the support.

Most preferably, a single probe molecule 104 is immobilized on a singlemicroparticle 103. However, two or more probe molecules may beimmobilized on a single microparticle as illustrated in FIG. 3.

When the probe molecule is a short nucleic acid sample fragment, onlymicroparticles each bearing a single probe molecule bound thereto can beselectively obtained after binding the probe molecule to themicroparticles. Typically, when microparticles in a number ten times thenumber of probe molecules were allowed to react with each other, about90% of microparticles did not capture a probe molecule, but about 9% ofmicroparticles captured each a single probe molecule. This result is ingood agreement with a predicted result on the assumption of Poissondistribution. Accordingly, when only microparticles capturing a probemolecule are collected, 90% or more of the collected microparticles aremicroparticles each capturing only one molecule of probe molecule. Themicroparticles in this state may be subjected to a process such asseparation by molecular weight, collection with magnetic microparticles,or electrophoretic separation using difference in electric charge. Thisgives microparticles each bearing a single molecule of probe moleculewith a higher purity.

Bonding pads 102 may be formed on a flat, smooth support 101 by using athin film processing which has been practically used in semiconductortechnologies. Typically, bonding pads 102 can be prepared by vapordeposition/sputtering through a mask, or by vapor deposition/sputteringto form a thin film and dry or wet etching of the thin film. Regularalignment of bonding pads 102 can be readily achieved using such thinfilm processing. The distance between pads can be appropriatelyadjusted. When light detection is performed using a detector, thedistance between pads is preferably 500 nm or more in view of thediffraction limit of light detection.

There are various possible ways to detect information related to nucleicacid samples in the nucleic acid analysis device according to the firstembodiment. In view of sensitivity and convenience, a method involvingfluorescence detection is preferably used. In this case, initially,nucleic acid samples may be supplied to the nucleic acid analysis deviceso as to allow probe molecules 104 to capture the nucleic acid samples.Next, nucleotides each having a fluorescent dye are supplied thereto.When the probe molecules 104 are DNA probes, a nucleic acid synthetasemay be supplied. Nucleic acid elongation may be induced on the device,followed by detection of fluorescence emitted from the fluorescent dyeincorporated into nucleic acid chains during the elongation. In thiscase, a so-called sequential elongation technique can be readilyachieved by repeating the steps of supplying one type of nucleotide,washing unreacted nucleotides, observing fluorescent emissions, andsupplying another type of nucleotide. After observation of fluorescentemissions, fluorescence from the fluorescent dye may be quenched, or anucleotide having a fluorescent dye at a phosphate moiety may be used toinduce a continuous reaction. Thus, information on the nucleotidesequences of nucleic acid samples can be obtained.

Alternatively, four types of nucleotides having different fluorescentdyes may be supplied and a continuous nucleic acid elongation may beinduced without washing, followed by continuous observation offluorescent emissions. Thus, a so-called real-time reaction process canbe realized. In this case, when a nucleotide having a fluorescent dye ata phosphate moiety is used, the phosphate moiety may be cleaved afterelongation, and this enables continuous fluorescent detections withoutquenching, to obtain information on the nucleotide sequences of nucleicacid samples.

Reasons why a single microparticle 201 is to be immobilized on a bondingpad 202 will be described with reference to FIGS. 4A and 4B. When themicroparticles 201 are to be immobilized on bonding pads 202, two ormore microparticles 201 could be immobilized on a single bonding pad202. If two or more microparticles 201 are immobilized thereon,information from different types of nucleic acid fragments areoverlapping, and this may impede accurate nucleic acid analysis.Therefore, a single microparticle 201 should be immobilized on a singlebonding pad 202. A bonding pad, if having a large diameter L and therebyhaving a large area as illustrated in FIG. 4A, could capture twomicroparticles. A bonding pad, if having a large thickness, could adsorbtwo microparticles and thereby capture two microparticles as illustratedin FIG. 4B, even when the bonding pad has a small diameter L1.

To avoid these, the present inventors repeated immobilizationexperiments under various conditions and made intensive investigations.As a result, they have found that immobilization of a singlemicroparticle 201 to a single bonding pad 202 is achieved when thebonding pad 202 has a diameter L of equal to or less than the diameter Dof the microparticle 201, namely, the ratio of the diameter 2D to thediameter L is equal to or more than 1; and when the surface of thesupport other than bonding pads is covered with a film for thesuppression of non-specific adsorption. Although microparticles repelone another, a single bonding pad, if having an apparent diameter largerthan twice that of a microparticle, could capture two or moremicroparticles even when the film for the suppression of non-specificadsorption is provided in combination. A device illustrated in FIG. 4Cincludes a bonding pad having an apparent diameter L₂ smaller than theapparent diameter D of a microparticle but having a large thickness t₁.This device does not employ the film suppressing non-specificadsorption. In this case, the bonding pad could capture microparticleson its lateral side, as illustrated in FIG. 4B.

When a microparticle 201 having a diameter D equal to or more than thediameter L of a bonding pad 102 is immobilized on the bonding pad 102,unreacted linear molecules may be covered with the immobilizedmicroparticle 201 and prevented from reacting with another microparticle201.

In consideration of this, a bonding pad preferably has a thickness (t₂)as small as possible. This is because, if a bonding pad 202 has a largethickness and thereby has a large lateral-side area, two or moremicroparticles 201 could be immobilized on a single bonding pad 202 evenwhen the bonding pad 202 has a diameter equal to or less than thediameter of the microparticles 201.

However, according to an embodiment of the present invention,immobilization of two or more microparticles 201 to a single bonding pad202 can be prevented even when the bonding pad 202 has a relativelylarge thickness. This is achieved by covering the lateral side of thebonding pad 202 with an organic polymer 204, such as a PEG (polyethyleneglycol), which suppresses adsorption of a biomolecule, as illustrated inFIGS. 5 and 6. This eliminates the influence of the thickness of bondingpad on the number of microparticles to be immobilized. The presentinvention therefore enables easy control of the thicknesses of bondingpads upon formation and provides a higher production yield.

A process of covering the lateral side of a bonding pad 202 with a PEGusing a silane coupling agent in a nucleic acid analysis device will bedescribed. This process employs a PEG-silane agent prepared bypolymerization of a PEG with a silane coupling agent. The PEG-silaneagent for use herein is typified by2-[methoxy(polyethylene-oxy)propyl]-trimethoxysilane (Gelest, Inc.). APEG-silane film having a thickness equivalent to the thickness of abonding pad 202 is formed in the nucleic acid analysis device. Asingle-layer PEG-silane film has a thickness of about 1 nm. When thisthickness is less than the thickness of the bonding pad 202, amultilayered PEG-silane film may be prepared so as to have a thicknessequivalent to the thickness of the bonding pad 202. The film wasprepared in the following manner. The PEG-silane agent was dissolved ina solvent to give a solution, a catalyst such as triethylamine was addedto the solution to give a mixture, and the nucleic acid analysis devicewas immersed in the mixture at 60° C. for one hour.

The nucleic acid analysis device was retrieved from the mixture andbaked in an electric furnace at 130° C. for one hour. A thickness of asilane film on the support was measured with a spectroscopicellipsometer. The measurement demonstrated the presence of a silane filmhaving a thickness of 14 nm. It was also demonstrated that the thicknessof film can be controlled in the range of 1 to 14 nm by modifyingreaction conditions such as the concentration of the PEG-silane agent,baking temperature, and baking time. A PEG-silane film having athickness of 10 nm was actually formed on a nucleic acid analysis devicehaving a bonding pad 202 with a thickness of 10 nm. As a result, thelateral side of the bonding pad 202 could be covered with the PEG-silanefilm. This was verified by observation of the cross section of thenucleic acid analysis device under a scanning electron microscope (SEM).

Bonding pads of some materials may be covered with the PEG-silane filmalso on the top side. In this case, the binding of the PEG-silane agentto the top side of a bonding pad can be prevented by covering thebonding pad 202 typically with a molecule capable of inhibiting silanoladsorption, prior to the treatment to form a PEG-silane film. Forexample, when the flat, smooth support 301 is made of a quartz glass andthe bonding pad 202 is made of titanium oxide, a poly(vinylphosphoricacid) (PVPA) can be used as the molecule capable of inhibiting silanoladsorption, to cover titanium oxide alone. Such poly(vinylphosphoricacid) (PVPA) is adsorbed on titanium oxide but is not adsorbed on quartzglass.

Only a PEG-silane film present on the top side of a bonding pad can beremoved according to the difference in adsorption power of thePEG-silane film, even when the molecule capable of inhibiting silanoladsorption is not used. Specifically, a PEG-silane film is more weaklyadsorbed on a metal or metal oxide constituting a bonding pad than on aquartz glass or sapphire constituting a flat, smooth support 301, andonly a PEG-silane film present on the bonding pad can be removed by acleaning step using ultrasound or a surfactant. The present inventorsactually verified that, when a PEG-silane film was prepared so as tohave a thickness equivalent to that of a bonding pad 202, only thelateral side of the bonding pad 202 could be fully covered with thePEG-silane film by covering the prepared bonding pad 202 with a moleculecapable of inhibiting silanol adsorption before the formation of thePEG-silane film, or by removing the formed PEG-silane film throughcleaning. The resulting nucleic acid analysis device including thePEG-silane film fully covering only the lateral side of the bonding pad202 was allowed to react with a microparticle 201 whose surface had beenmodified with avidin. As a result, the microparticle 201 was immobilizedonto the top side of the bonding pad 202 but was not immobilized to thelateral side thereof. This was verified through observation under ascanning electron microscope (SEM).

As is described above, the organic polymer (film) 204 for preventingnon-specific adsorption could be easily prepared on a nucleic acidanalysis device. This enabled highly effective prevention ofnon-specific adsorption and indicated significant noise reduction. Thisalso remarkably improved the percentage of immobilization ofmicroparticles 201 to bonding pads 202 on a one-to-one basis. Theseimprovements help the nucleic acid analysis device to have a remarkablyhigher throughput.

Second Embodiment

Embodiments relating to a method for producing a nucleic acid analysisdevice will be illustrated with reference to FIGS. 7 and 8.

In an embodiment illustrated in FIG. 7, a film of a material forconstituting a bonding pad 305 is deposited on a flat, smooth support301 by sputtering (FIG. 7( b)) to form a thin metal film 302. Thematerial is typified by gold, titanium, nickel, or aluminum. When theflat, smooth support 301 is a glass support or sapphire support, andwhen the bonding pad 305 is to be formed from gold, aluminum, or nickel,a thin film of titanium or chromium is preferably deposited between thesupport and the bonding pad 305 so as to enhance adhesion between thematerial constituting the support and the material constituting thebonding pad 305.

A resist pattern 303 is formed on the thin metal film 302 (FIG. 7( c)).Next, the thin metal film 302 in a region other than the resist patternis removed by etching (FIG. 7( d)). The resist 303 is then removed tocomplete a bonding pad 305. Next, linear molecules 304 are allowed toreact with the flat, smooth support 301 (FIG. 7( e)). The linearmolecules 304 are not adsorbed on the flat, smooth support 301 butadsorbed on the bonding pad 305. When the bonding pad 305 is made fromgold, titanium, aluminum, or nickel, the linear molecules 304 eachpreferably have a sulfhydryl group, phosphoric group, or thiazole groupas a terminal functional group. The linear molecules may have biotin asa functional group. After the reaction of the linear molecules with theflat, smooth support 301, a thin film 306 is prepared on the surface ofthe flat, smooth support 301 except for a region where the bonding pad305 has been formed (FIG. 7( f)).

Next, a microparticle 103 is bound to the linear molecules 304 throughchemical bonds, and one or plural probe molecules are immobilized on thesurface of the microparticle (FIG. 7( g)).

The thin film 306 includes a material organic polymer for preventingnon-specific adsorption of the microparticle 103. The type of theorganic polymer for preventing non-specific adsorption is suitablyselected depending on the surface condition of the microparticle 103. Anegatively-charged organic polymer is selected when the microparticle103 has a negatively charged surface, so as to repel each other. Ahydrophobic organic polymer is selected when the microparticle 103 has ahydrophilic surface; whereas a hydrophilic polymer is selected when themicroparticle 103 has a hydrophobic surface. Typically, a polyethyleneglycol (PEG), a polyacrylamide, or 3-glycidoxypropylmethoxysilane (GOPS)may be used as the organic polymer 306 for preventing non-specificadsorption when the microparticle 103 is one modified with hydrophilicavidin.

Another embodiment of a method for producing a nucleic acid analysisdevice will be illustrated with reference to FIG. 8. This method allowslinear molecules 403 to remain only on the top side of a bonding pad 406even when the linear molecule does not have selectivity between thebonding pad 406 and a flat, smooth support 401.

With reference to FIG. 8( b), the linear molecules 403 is allowed toreact before etching of a deposited thin metal film 402. In this case, apattern is formed with a resist 404 after the reaction of the linearmolecules 403 (FIG. 8( c)). The thin metal film 402, other than theregion protected by the resist 404, is removed together with the linearmolecules 403 by etching (FIG. 8( d)). Next, a thin film of an organicpolymer 405 for preventing non-specific adsorption is formed on theresist 404 and the flat, smooth support 401 (FIG. 8( e)).

The resist 404 is then stripped to thereby remove the resist 404together with the organic polymer 406 for preventing non-specificadsorption formed on the resist 404. Thus, a bonding pad bound to apatterned linear molecule 403 is formed (FIG. 8( f)). A microparticle103 is then bound to the linear molecules 403 through chemical bonds,and one or plural probe molecules are immobilized on the surface of themicroparticle (FIG. 8( g)).

The microparticles have preferably been modified with avidin on surface.When gold or platinum microparticles are used, modification with avidincan be easily performed by allowing the microparticles to reactsequentially with aminothiol, biotin-succinimide (NHS-Biotin; PierceBiotechnology, Inc.), and streptavidin in this order. Whenmicroparticles of another metal than gold and platinum are used, surfacemodification of the metal microparticles with avidin can be easilyperformed by subjecting the microparticles to heating in an oxygenatmosphere to oxidize the surface, and allowing the surface-oxidizedmicroparticles to react sequentially with aminosilane,biotin-succinimide (NHS-Biotin; Pierce Biotechnology, Inc.), andstreptavidin in this order.

The each of the microparticles bearing a nucleic-acid-capturing probeimmobilized thereon are then allowed to react with the flat, smoothsupport 401 and thereby yields a nucleic acid analysis device accordingto the second embodiment.

Third Embodiment

In the third embodiment, an exemplary preferred configuration of anucleic acid analyzer using a nucleic acid analysis device will beillustrated with reference to FIG. 9. The nucleic acid analyzeraccording to the third embodiment includes a nucleic acid analysisdevice; a supplier for supplying a nucleic acid synthetase, a nucleicacid sample, and a nucleotide having a fluorescent dye to the nucleicacid analysis device; an irradiator for irradiating the nucleic acidanalysis device with light; and an emission detector for detectingfluorescence emitted from the fluorescent dye incorporated into anucleic acid chain through nucleic acid elongation that is induced bythe coexistence of a nucleotide, a nucleic acid synthetase, and anucleic acid sample on the nucleic acid analysis device. Morespecifically, the device 505 is placed in a reaction chamber whichincludes a cover plate 501, a detection window 502, an inlet 503, and anoutlet 504, which inlet and outlet serve as solution exchange ports. Apolydimethylsiloxane (PDMS) may be used as a material for the coverplate 501 and the detection window 502.

The detection window 502 may have a thickness of 0.17 mm. Ayttrium-aluminum-garnet (YAG) laser source (wavelength: 532 nm; output20 mW) 507 and a YAG laser source (wavelength: 355 nm; output: 20 mW)508 emit laser beams 509 and 510, respectively. The laser beam 509 aloneis circularly polarized using a quarter-wave plate (λ/4 plate) 511 so asto adjust the two laser beams concentrically with a dichroic mirror 512(for reflecting light with a wavelength of 410 nm or less), followed bylight condensing using a lens 513. Then, the device 505 is irradiatedwith the light (laser beam) via a prism 514 at an angle equal to orlarger than the relevant critical angle.

An example in which gold microparticles each having a diameter ofapproximately 50 nm are used as microparticles is described below.

In this case, localized surface plasmon is generated on goldmicroparticles present on the surface of a device 505 via laserirradiation. Accordingly, a fluorophore of a target substance capturedby a DNA probe bound to a gold microparticle is present in the enhancedelectric field. A fluorophore is excited with laser light, and theenhanced fluorescent emission is partially output through the detectionwindow 502. A parallel light beam is formed with fluorescence passedthrough the detection window 502 using an objective lens 515 (x60;NA=1.35; operating distance: 0.15 mm). Background light and excitationlight are then intercepted by an optical filter 516, resulting inimaging with a two-dimensional CCD camera 518 via an imaging lens 517.

When a sequential reaction system is employed, an exemplary nucleotidehaving a fluorescent dye usable herein is a nucleotide including a3′-O-allyl group is added as a protective group at the 3′ OH position ofribose moiety; and a fluorescent dye bound via an allyl group at the5-position of pyrimidine or the 7-position of purine, as disclosed inNPL 5. The allyl group may be cleaved by light irradiation (at awavelength typically of 355 nm) or by contact with palladium. Thisenables both quenching of light emitted from a dye and control ofelongation. Even in the case of a sequential reaction, there is no needto remove unreacted nucleotides by cleaning. Additionally, real-timemeasurement during elongation is also possible, because a cleaning stepis not necessary. In this case, there is no need to add a 3′-O-allylgroup as a protective group at the 3′ OH position of ribose moiety inthe nucleotide. A nucleotide bound to a dye via a functional groupcapable of being cleaved by light irradiation (at a wavelength typicallyof 355 nm) may be used.

When semiconductor microparticles are used as the microparticles, theabove example of a nucleic acid analyzer can also be applied. Forexample, when a Qdot® 565 (Invitrogen (Life Technologies Corporation))is used as a semiconductor microparticle, sufficient excitation can beinduced using a YAG laser source 307 (wavelength: 532 nm; output: 20mW). When the excitation energy is transferred to Alexa Fluor® 633(Invitrogen (Life Technologies Corporation)) that is not excited withlight at a wavelength of 532 nm, fluorescence emission occurs.Specifically, a dye bound to an unreacted nucleotide is not excited.Only after a nucleotide bound to a dye is captured by a DNA probe andthus becomes in proximity to a semiconductor microparticle, whichresults in energy transfer, light is emitted from the dye. Capturednucleotides can therefore be identified by fluorometry.

Microparticles made from an inorganic polymer or organic polymer are notexcited even upon irradiation with light from an external light source.For this reason, light emission from a fluorescent dye due to transferof excitation energy does not occur, whereas unreacted nucleotides alsoemit light, and this may cause noise. However, incorporated nucleotidesalone can be allowed to emit light by binding a nucleic acid synthetaseto a microparticle capable of inducing energy transfer, such as asemiconductor microparticle. Alternatively, fluorescence from suchincorporated nucleotides can be enhanced by binding gold, silver,platinum, or aluminum to a nucleic acid synthetase. Fluorescence arounda metal pad for immobilizing a microparticle can be enhanced, andthereby the signal-to-noise ratio can be increased by using gold,silver, platinum, or aluminum as a material for constituting the metalpad.

As is described above, when a nucleic acid analyzer is assembled usingthe nucleic acid analysis device according to the third embodiment,analysis time can be shortened without introducing a cleaning step intothe analysis process, and the device and the analyzer can be simplified.Accordingly, not only measurement based on a sequential reaction systembut also real-time measurement can be achieved during nucleotideelongation. This provides significant throughput improvement overcustomary techniques.

According to the present invention, many types of nucleic acid fragmentscan be regularly aligned at a high density and immobilized on a supportby the medium of microparticles. This allows immobilization of a singlemolecule of nucleic acid sample fragment at a high percentage merely bya simple treatment of the support without increasing the number of stepsin production process of a nucleic acid analysis device; and alsoenables noise reduction by suppression of non-specific adsorption ofmicroparticles. This in turn enables low-cost and high-throughputanalysis of nucleic acid samples.

REFERENCE SIGNS LIST

-   -   101, 203, 301, 401 flat, smooth support    -   102, 202, 305 bonding pad    -   103, 201 microparticle    -   104 probe molecule    -   105 linear molecule    -   106, 107, 205, 206, 304, 403 terminal functional group of linear        molecule    -   108, 204, 306, 405 organic polymer for preventing non-specific        adsorption    -   302, 402 thin metal film    -   303, 404 resist    -   501 cover plate    -   502 detection window    -   503 inlet    -   504 outlet    -   505 nucleic acid analysis device    -   506 flow channel    -   507, 508 YAG laser source    -   509, 510 laser beam    -   511 quarter-wave plate    -   512 dichroic mirror    -   513 lens    -   514 prism    -   515 objective lens    -   516 optical filter    -   517 imaging lens    -   518 two-dimensional CCD camera

1. A nucleic acid analysis device, comprising: a support; a plurality ofbonding pads arranged on a surface of the support; a thin-film layercovering the surface of the support in a region other than the bondingpads; microparticles, where a single microparticle is bound to each ofthe bonding pad; and a probe molecule or molecules of a single typeimmobilized on each of the microparticle, wherein the microparticles arebound to the bonding pads through chemical bonds, and wherein thethin-film layer is capable of suppressing non-specific adsorption of themicroparticles on the surface of the support.
 2. The nucleic acidanalysis device of claim 1, wherein a single probe molecule isimmobilized on a single microparticle.
 3. The nucleic acid analysisdevice of claim 1, wherein the probe molecule or molecules comprise anucleic acid or a nucleic acid synthetase.
 4. The nucleic acid analysisdevice of claim 1, wherein the microparticles is a material selectedfrom the group consisting of semiconductors, metals, inorganic polymers,and organic polymers.
 5. The nucleic acid analysis device of claim 1,wherein the bonding pads comprise a material selected from the groupconsisting of gold, titanium, nickel, and aluminum.
 6. A method forproducing a nucleic acid analysis device which comprises the steps of:forming a thin metal film on a surface of a support; selectively etchingthe thin metal film to form a plurality of bonding pads; introducing alinear molecule into each of the bonding pad, the linear moleculecapable of being adsorbed on the bonding pad; binding an each of themicroparticles to the linear molecule through a chemical bond; andimmobilizing a probe molecule or molecules to each of the microparticlethrough a chemical bond.
 7. The method for producing a nucleic acidanalysis device of claim 6, wherein the bonding pads are regularlyarranged on the support.
 8. The method for producing a nucleic acidanalysis device of claim 6, wherein two or more probe molecules areimmobilized on a single microparticle.
 9. The method for producing anucleic acid analysis device of claim 6, wherein the bonding pads havediameters each twice or less the diameters of the microparticles.
 10. Amethod for producing a nucleic acid analysis device which comprises thesteps of: forming a thin metal film on a surface of a support;introducing a linear molecule film onto the thin metal film, the linearmolecule film capable of being adsorbed on the thin metal film; forminga plurality of dummy bonding pads, selectively etching the the linearmolecule film and thin metal film, removing the dummy bonding pads toexpose the linear molecule film, and thereby forming a plurality ofbonding pads each having the thin metal film and the linear moleculefilm; binding each of the microparticles to the exposed linear moleculefilm through a chemical bond; and immobilizing a probe molecule ormolecules to each of the microparticles through a chemical bond.
 11. Themethod for producing a nucleic acid analysis device of claim 10, whereinthe bonding pads are regularly arranged on the support.
 12. The methodfor producing a nucleic acid analysis device of claim 10, wherein two ormore probe molecules are immobilized on a single microparticle.
 13. Themethod for producing a nucleic acid analysis device of claim 10, whereinthe bonding pads have diameters each twice or less the diameters of themicroparticles.
 14. A nucleic acid analyzer which comprises: a nucleicacid analysis device including a support and microparticles regularlyimmobilized on a surface of the support, each of the microparticleshaving a probe molecule or molecules capable of capturing a nucleic acidto be analyzed; a supplier for supplying a nucleic acid sample and anucleotide having a fluorescent dye to the nucleic acid analysis device;an irradiator for irradiating the nucleic acid analysis device withlight; and an emission detector for detecting fluorescence emitted fromthe fluorescent dye incorporated into a nucleic acid chain throughnucleic acid elongation induced by the coexistence of a nucleotide, anucleic acid synthetase, and a nucleic acid sample; wherein the nucleicacid analyzer obtains information on nucleotide sequence of the nucleicacid sample, and wherein the nucleic acid analysis device furthercomprises a plurality of bonding pads arranged on the surface of thesupport at positions where the microparticles are immobilized, themicroparticles are bound to the bonding pads through chemical bonds, andthe nucleic acid analyzer further comprises a linear molecule filmcovering the support in a region other than the bonding pads.
 15. Thenucleic acid analyzer of claim 14, wherein a single probe molecule isimmobilized on a single microparticle.
 16. The nucleic acid analyzer ofclaim 14, wherein the probe molecule or molecules comprise a nucleicacid or a nucleic acid synthetase.
 17. The nucleic acid analyzer ofclaim 14, wherein the microparticles comprise a material selected fromthe group consisting of semiconductors, metals, inorganic polymers, andorganic polymers.
 18. The nucleic acid analyzer of claim 14, the bondingpads comprise a material selected from the group consisting of gold,titanium, nickel, and aluminum.